Sphingosine-1-phosphate (S1P) has been shown to regulate cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) expression and IL-6 secretion in various respiratory diseases. However, the mechanisms underlying S1P-induced COX-2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX-2 expression. S1P also induced PGE2 and IL-6 secretion which were reduced by the inhibitors of COX-2 (NS-398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, PYK2, p38, p42, JNK2, c-Jun, or c-Fos reduced S1P-induced COX-2 expression and PGE2 /IL-6 secretion. Moreover, S1P induced c-Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c-Jun phosphorylation. We observed that S1P-induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c-Src/PYK2-dependent pathway. S1P also enhanced c-Fos, but not c-Jun mRNA and protein expression and the AP-1 promoter activity. S1P-induced c-Fos mRNA and protein expression, c-Jun phosphorylation, and AP-1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P-induced COX-2 expression and PGE2 /IL-6 generation was mediated through S1PR1/3/c-Src/PYK2/p42/p44 MAPK- or JNK1/2- and S1PR1/3/c-Src/p38 MAPK-dependent AP-1 activation.

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation.

Up-regulation of cytosolic phospholipase A(2) (cPLA(2)) by cigarette smoke extract (CSE) may play a critical role in airway inflammatory diseases. However, the mechanisms underlying CSE-induced cPLA(2) expression in human tracheal smooth muscle cells (HTSMCs) were not completely understood. Here, we demonstrated that CSE-induced cPLA(2) protein and mRNA expression was inhibited by pretreatment with the inhibitors of AP-1 (tanshinone IIA) and p300 (garcinol) or transfection with siRNAs of c-Jun, c-Fos, and p300. Moreover, CSE also induced c-Jun and c-Fos expression, which were inhibited by pretreatment with the inhibitors of NADPH oxidase (diphenyleneiodonium chloride and apocynin) and the ROS scavenger (N-acetyl-L-cysteine) or transfection with siRNAs of p47(phox) and NADPH oxidase (NOX)2. CSE-induced c-Fos expression was inhibited by pretreatment with the inhibitors of MEK1 (U0126) and p38 MAPK (SB202190) or transfection with siRNAs of p42 and p38. CSE-induced c-Jun expression and phosphorylation were inhibited by pretreatment with the inhibitor of JNK1/2 (SP600125) or transfection with JNK2 siRNA. CSE-stimulated p300 phosphorylation was inhibited by pretreatment with the inhibitors of NADPH oxidase and JNK1/2. Furthermore, CSE-induced p300 and c-Jun complex formation was inhibited by pretreatment with diphenyleneiodonium chloride, apocynin, N-acetyl-L-cysteine or SP600125. These results demonstrated that CSE-induced cPLA(2) expression was mediated through NOX2-dependent p42/p44 MAPK and p38 MAPK/c-Fos and JNK1/2/c-Jun/p300 pathways in HTSMCs.

Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)-1, displays an anti-inflammatory effect in various cellular injuries, but the precise mechanisms of HO-1 expression remain unknown. We used the transition metal carbonyl compound carbon monoxide-releasing molecule-2 (CORM-2) that acts as carbon monoxide donor. The effects of CORM-2 on expression of HO-1 in human tracheal smooth muscle cells (HTSMCs) were determined by Western blot, real-time PCR, and promoter activity assay. In HTSMCs, CORM-2 activated Nrf2 through the activation of a c-Src/EGFR/PI3K/Akt-dependent pathway, resulting in HO-1 expression. We showed that CORM-2-induced HO-1 protein and mRNA levels were inhibited by the inhibitor of c-Src (PP1 or SU6656), EGFR (AG1478), PI3K (LY294002), Akt (SH-5), JNK1/2 (SP600125), or p38 MAPK (SB202190) and transfection with siRNA of c-Src, EGFR, Akt, p38, JNK2, or Nrf2 in HTSMCs. We also showed that CORM-2 stimulated c-Src, EGFR, Akt, p38 MAPK, and JNK1/2 phosphorylation. CORM-2 also enhanced Nrf2 translocation from the cytosol to the nucleus and antioxidant response element (ARE) promoter activity. Moreover, CORM-2 mediated p38 MAPK and JNK1/2 activation via a c-Src/EGFR/PI3K/Akt pathway, which further enhanced Nrf2 activation and translocation. Finally, we observed that CORM-2 induced in vivo binding of Nrf2 to the HO-1 promoter. CORM-2 activates the c-Src/EGFR/PI3K/Akt/JNK1/2 and p38 MAPK pathways, which in turn trigger Nrf2 activation and ultimately induces HO-1 expression in HTSMCs. Thus, the HO-1/CO system might be potential therapeutics in airway diseases.

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.